Campylobacter jejuni is a prevalent gastrointestinal pathogen associated with increasing rates of antimicrobial resistance development. It was also the first bacterium demonstrated to possess a general N-linked protein glycosylation pathway capable of modifying > 80 different proteins, including the primary Campylobacter multidrug efflux pump, CmeABC. Here we demonstrate that N-glycosylation is necessary for the function of the efflux pump and may, in part, explain the evolutionary pressure to maintain this protein modification system. Mutants of cmeA in two common wildtype (WT) strains are highly susceptible to erythromycin (EM), ciprofloxacin and bile salts when compared to the isogenic parental strains. Complementation of the cmeA mutants with the native cmeA allele restores the WT phenotype, whereas expression of a cmeA allele with point mutations in both N-glycosylation sites is comparable to the cmeA mutants. Moreover, loss of CmeA glycosylation leads to reduced chicken colonization levels similar to the cmeA knock-out strain, while complementation fully restores colonization. Reconstitution of C. jejuni CmeABC into Escherichia coli together with the C. jejuni N-glycosylation pathway increases the EM minimum inhibitory concentration and decreases ethidium bromide accumulation when compared to cells lacking the pathway. Molecular dynamics simulations reveal that the protein structures of the glycosylated and non-glycosylated CmeA models do not vary from one another, and in vitro studies show no change in CmeA multimerization or peptidoglycan association. Therefore, we conclude that N-glycosylation has a broader influence on CmeABC function most likely playing a role in complex stability.

中文翻译：

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CmeABC多药外排泵的N-糖基化对于空肠弯曲杆菌的最佳功能是必需的。

空肠弯曲菌是一种常见的胃肠道病原体，与抗菌素耐药性发展的速率增加有关。它也是第一个被证明具有一般的N联蛋白糖基化途径的细菌，该途径能够修饰> 80种不同的蛋白，包括初级弯曲杆菌多药外排泵CmeABC。在这里，我们证明了N-糖基化对于外排泵的功能是必需的，并且可以部分解释维持该蛋白质修饰系统的进化压力。与同基因的亲本菌株相比，两种常见的野生型（WT）菌株中cmeA突变体对红霉素（EM），环丙沙星和胆汁盐高度敏感。cmeA突变体与天然cmeA等位基因的互补可恢复WT表型，而在两个N-糖基化位点均具有点突变的cmeA等位基因的表达与cmeA突变体相当。此外，CmeA糖基化作用的丧失导致鸡定植水平降低，类似于cmeA敲除菌株，而互补作用则完全恢复了定植。与缺少空肠弯曲杆菌CmeABC的细胞相比，空肠弯曲杆菌CmeABC与空肠弯曲杆菌N-糖基化途径的重组增加了EM最低抑制浓度并减少了溴化乙锭的积累。分子动力学模拟表明，糖基化和非糖基化的CmeA模型的蛋白质结构彼此之间没有变化，并且体外研究表明CmeA的多聚化或肽聚糖结合没有变化。所以，